aab bioflux · cv. pacovan) by an am fungi (glomus clarum) improved the dry weight of root (80%),...

AAB Bioflux, 2009, Volume 1, Issue 2. http://www.aab.bioflux.com.ro

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AAB BIOFLUX Advances in Agriculture & Botanics- International Journal of the Bioflux Society Water relationships and agronomic indices of sunflower infection by microbial inoculants under saline condition 1Mostafa Shirmardi, 1Gholam R. Savaghebi, 2Kazem Khavazi, 1Ali Akbarzadeh, 1Mohsen Farahbakhsh, 2Farhad Rejali, and 1Abdolvahab Sadat

1 Department of Soil Science, Faculty of Water & Soil Engineering, University College of Agriculture & Natural Resources, University of Tehran, Karaj, Iran; 2 Soil & Water

Research Institute, Tehran, Iran. Corresponding author: Ali Akbarzadeh, [email protected]

Abstract. Salinity is one of the most important limiting factors for plant growth and crop production. Saline soils with having low activity in nutrient elements and high osmotic pressure in solution phase make so much nutrition problems and physiological drought in plant. In this study, the effects of arbuscular mycorrhiza fungi and plant growth promoting bacteria with ability to produce ACC deaminase enzyme on water relationships and agronomic indices of sunflower cultivars were investigated. The saline soil (EC = 7.6 dS m-1) used in this investigation was taken from Karaj region (Eshtehard) of Iran. In a factorial experimental design on the basis of randomized complete block samples, three levels of arbuscular mycorrhizal inoculants (non inoculation, inoculation with Glomus etunicatum and Glomus intradices), four levels of Pseudomonas fluorescens inoculants (non inoculation, inoculation with Pseudomonas fluorescens strains 4, 9 and 12) on two cultivars of sunflower (Euroflor and Master) with four replications per treatment were applied. Results showed that all of the treatments significantly (P < 0.05) increased relative water content (RWC) of two cultivars. Single inoculation of Pseudomonas fluorescens strains 12 and Glomus etunicatum and also co-inoculation of fungi with each one of the bacteria enhanced fresh and dry weight of tray in Euroflor cultivar, compared with the control treatment. In addition, all of the treatments except Pseudomonas fluorescens strains 4 and also Glomus intradices significantly (P < 0.05) increased dry weight of above ground part in Euroflor cultivar. Key Words: Salinity, Plant growth promoting rhizobacteria, Arbuscular mycorrhiza fungi, Sunflower.


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Introduction. The environmental stress of salinity reduces growth and agricultural productivity more than any other factors (Karakas et al 1997). The direct effects of salt on plant growth may involve (1) a reduction in the osmotic potential of the soil solution that reduces plant-available water, and (2) toxicity of excessive Na+ or Cl– towards the plasma membrane. Osmotic effects are associated with inhibition of cell wall extension and cellular expansion, leading to reduced plant growth (Feng et al 2002). Plants being immobile cannot evade salt stress in the same way as mobile organisms. So, they show many morphological and physiological alterations to acclimatize to unfavorable environment (Sakamoto & Murata 2002). For reducing of adverse effects of soil salinity there are several methods. One of these methods is using of biological fertilizers. Due to many bioenvironmental problems caused by chemical fertilizers, these days many farmers tend to use biological fertilizers for achievement to sustainable agriculture. Plant growth-promoting rhizobacteria (PGPR) and arbuscular mycorrhizal (AM) fungi represent two main groups of beneficial microorganisms of the rhizosphere which known as biological fertilizers (Russo et al 2005). The beneficial effect of PGPRs as well as AM fungi on plants is well documented (Gamalero et al 2003). Sunflower (Helianthus annuus L.) is an important oilseed crop grown in different parts of the world. It has C3 photosynthetic pathway and is mostly cultivated in arid and semiarid regions (Iqbal et al 2005). Although many studies on salt tolerance of sunflower has been carried out, basic research on role of microbial inoculants in salt tolerance of sunflower is scarce. Most investigations regarding PGPRs and AM fungi efficiency in increasing plant tolerance to salinity are related to other agricultural crops. However, in all of these investigations it is clear that reducing salinity-induced ethylene by any mechanism can decrease the negative impact of salinity onto plant growth. In another word, plant inoculation with PGPR containing ACC deaminase activity is so helpful in sustaining plant growth and development under stress conditions by reducing stress-induced ethylene production (Saleem et al 2007). Mayak et al (2004) evaluated the role of a PGPR (Achromobacter piechaudii) in resistance of tomato plant to salt stress in dry salty environments of Israel. They reported that this bacterium significantly increased the fresh and dry weights of tomato seedlings grown in the presence of up to 172 mM NaCl salt. Also, in the presence of salt the bacterium increased the water use efficiency (WUE). They suggested that the bacterium act to alleviate the salt suppression of photosynthesis. Similarly, Hamdia et al (2004) studied the role of a PGPR (Azospirillum brasilense) in enhancing of corn growth at saline conditions. They reported that inoculating of corns varieties that were sensitive to salt stress had significant effect on increasing their dry root and shoot yield as compared with the control treatments. In addition, Saravanakumar and Samiyappan (2007) reported that Pseudomonas Xuorescens strain TDK1 containing ACC deaminase activity enhanced the saline resistance in groundnut plants and increased yield as compared with that inoculated with Pseudomonas strains lacking ACC deaminase activity. Cheng et al (2007) have also pointed out that ACC deaminase bacteria conferred salt tolerance onto plants by lowering the synthesis of salt-induced stress ethylene and promoted the growth of canola in saline environment. Yano-Melo et al (2003) showed that inoculating of banana plants (Musa sp. cv. Pacovan) by an AM fungi (Glomus clarum) improved the dry weight of root (80%), shoot (83%), and the total leaf area (60%) compared to non-inoculated plants. The salt tolerance of banana as measured by leaf number and plant height increased considerably in presence of Glomus isolates. They suggested that inoculation with specific AM fungi therefore constitutes an alternative method to reduce banana plant stress caused by soil salinization. In addition, Al-Karaki (2006) indicated that pre-inoculation of tomato transplants with AM fungi improved yield and can help alleviate deleterious effects of salt stress on crop yield. He showed that pre-inoculated tomato plants with AM fungi irrigated with both saline and nonsaline water had greater shoot and root dry matter (DM) yield and fruit fresh yield than nonAM plants. The enhancement in fruit fresh yield due to AM fungi inoculation was 29% under nonsaline and 60% under saline water conditions. Also these results indicated that pre-inoculation of tomato transplants with AM fungi improved yield and could help alleviate deleterious effects of salt stress on crop yield. Sannazzaro et al (2006) found that AM fungi (Glomus intraradices) improved growth of L. glaber


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plants under saline conditions. They showed that mycorrhizal plants had higher values of net growth, shoot/root and protein concentrations than controls. Tolerant AM plants also showed higher chlorophyll levels than non-AM ones. Feng et al (2002) observed that the concentrations of chlorophyll were higher than in non-mycorrhizal plants compared with mycorrhizal plants under salty environments. However, mycorrhizal plants maintained higher root and shoot dry weights. The study of the antagonic or synergic effects of the different microbial inoculants when co-inoculated is a crucial step in the development of effective host-microorganism combinations. Kohler et al (2007) studied the interactions between the inoculation of lettuce plants with an arbuscular mycorrhizal fungus (Glomus intraradices) and a plant growth-promoting rhizobacterium (Bacillus subtilis). They reported that this co-inoculation synergistically increased plant growth compared with singly inoculated (about 77% greater with respect to the control plants). Other similar results confirmed the advantages of co-inoculation with PGPRs and AM fungi in increasing of plant growth (Germida & Walley 1996; Toro et al 1997; Caravaca et al 2005). Drought and salinity are already widespread in many regions of Iran, and are expected to cause serious salinization of the most arable lands of this region. Also, nowadays Iran is so dependent to other countries in the case of oily seeds. The cost of imports for edible oils or oily seed at Iran is more than 800 million dollar per year. This figure indicates the vital role of sunflower as an industrial crop having main source of high quality edible oil in Iran’s economy. Agro climatic condition of the most parts of Iran is such type that this crop can be grown successfully. But according to present information provided from Iranian soil’s map about 44.5 million hectare of soils in Iran have large amount of different salts (Banaei 2001). Hence, despite the economic importance of sunflower plant in Iran, there is limited study on the improvement of its tolerance, growth, yield and other its agronomic indices to the infecting by microbial inoculants under saline condition. This study was therefore carried out with three main objectives: (a) to investigate the effect of PGPRs (Strains of Pseudomonas fluorescens) and AMF (Glomus etunicatum and Glomus intradices) on reducing salt stress and improvement of growth, yield and other agronomic indices of sunflower plants, (b) to study the role of co-inoculation of sunflower plant by PGPRs and AMF in increasing its agronomic indices and (c) to evaluate and compare the growth response of two cultivars of sunflower (Euroflor and Master) to microbial inoculation. Material and Method. This study was carried out at greenhouse condition. Plastic pots having 22 cm height and 20 cm opening mouth diameter were selected for planting of sunflowers. The weight of each vacant pot was about 250 g. Approximately 4 kg of air-dried soil passed through a 4.8 mm sieve was added to each plastic pot. Two cultivars of sunflower seeds (Euroflor and Master) were provided from institute of seed and seedling of Karaj for this research. Before sowing the seeds into the pots, the seeds that their shape was similar were selected and then, were kept in the trays containing javellewater (2.5%) for 7 minute. Then the seeds were washed 6-7 times by distilled water and were held several days on the sterile papers inside the incubator for germination. Microbial inoculants including micorrhizal fungi and bacteria were prepared from

soil & water research institute of Iran as powdery forms and separated boxes. The mycorrhizal inoculants including Glomus etunicatum and Glomus intradices were isolated from saline soils of Tabriz plane which located in northern Iran. The fungi population in these inoculants was about 1.6×104 fungus per 1 g soil. The bacterial inoculants were consisted of strains 4, 9, 12 of Pseudomonas fluorescens (Table 1).

Before transferring the seedlings into the plastic pots, the soil was irrigated to the field capacity level. When the soil moisture was suitable for planting, according to the treatment design, in each pot 4 small holes were shaped and then, 2 g fungi inoculants along with 1 g bacterial inoculants were added these holes. After that in these entire holes, one seedling of sunflower was planted and pots were irrigated at the level of field capacity on the basis of the weight of each pot. According to soil analysis, the essential elements as chemical fertilizers were added to the soils. Just the phosphorous applied at the half of its optimum level due to proper studding the effect of micorrhizal fungi. After suitable establishing of plants in the plastic pots, the number of plant was reduced to two


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plants in each pot by removing them. The air temperature inside the greenhouse was hold approximately fixed at 25 ºC during the growth stages of sunflower plants. Also, the period of sunshine was about 12 hours throughout the plant growth. Approximately 90 days after transplanting, the sunflower plants were cut form their bottom. It was needed to a salty soil for doing the experiments. Therefore a pre-sampling performed from different locations in Karaj (Eshtehard) region of Iran. After determining the salinity of these pre-samples, the best place was selected. The sampling location was between 35°, 43′ in eastern latitude and 50°, 18′ in northern longitude. Silage maize is the major feeder crop followed by alfalfa and wheat are the main cereal crop in this region. Due to the lack of animal manure, crop production is based on mineral NPK fertilizers. This area is also situated in a river alluvial plain and the soil of this region belongs to xeric haplocambids. The soil texture is loamy with an average pH of 7.8, electrical conductivity (EC) of 7.6 dS m-1, organic matter (OM) content of 0.5%, CaCO3 content of 13.6%, gypsum content of 3.1%, total N content of 0.045, saturation percentage (SP) of 37.3%, sodium adsorption ratio (SAR) of 9.24, exchangeable sodium percentage (ESP) of 11% and cation exchange capacity (CEC) level of 16.9 meq per 100 g of soil. The levels of soluble Na, Mg, Ca, K, Cl and HCO3 in the soil were 45.6, 10.0, 39.7, 14.1, 46.5 and 5.3 meq L-1, respectively. Also the level of Fe, Zn, Cu and Mn extracted by DTPA in the soil were 2.8, 1.6, 1.3 and 10.0 mg kg-1, respectively.

Table 1

Some characteristics of bacteria in inoculants

Bacterium Activity of ACC deaminase (μmoles mg-1 h-1)

Auxin (μg ml-1)

Bacterial population (Cfu ml-1)

Pseudomonas fluorescence

strain 4 8.17 2.38 7.7×109


strain 9 4.45 0.93 2.1×109


strain 12 4.61 1.2 2.5×109

In this study the leave water potential was measured using a digital plant water potentiometer apparatus (EL540-300). Also, the plant chlorophyll was measured using a chlorophyll meter apparatus (SPAD-502) according to Fox et al (1994) method. For calculating the relative water content (RWC), the latest expanded plant’s leaves were cut and their weight measured. After that, the leaves soaked into the distilled water for 24 hours. After this saturation the leave surfaces was dried using a clean towel and their weights measured again. Ultimately the leaves were dried in the oven at 70 ºC temperature and the level of RWC calculated by following equation:

RWC = Fresh leaf weigh – Dry leaf weigh / Saturated leaf weigh – Dry leaf weigh (1) At the end of plant growth (90 day after transplanting) the leaf area was measured by means of a leaf area meter (GATE HOUSE). Also at this time other growth parameters including dry and fresh weight of above ground parts or organs (consisting of: leaf, stalk and tray), dry and fresh weight of trays, tray diameter, dry weigh of root and percentage of root colonization were also measured. For measuring the root colonization the Grindline Intersect method was used. In this method for segregation of roots from soil particles, the pot’s soil was saturated and then soil particles washed gently away by water. After clean-up the roots, 1 gr samples were taken from different parts of roots. These samples were maintained into vessels having a mixture of alcohol and water. For performance of colour-blending, the roots were heated at 90 ºC temperature into the 10% KOH solution for 1 hour. The washed roots were floated in a


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basic solution of 10% H2O2 for 20 minutes for doing the discolouration and then, the roots were washed for several times and soaked into a 1% HCl solution for 3 minutes for doing the acidification process. At the last stage, the roots transferred into the lactoglycerin-triphane blue for 48 hours for completion the coloration process (Philips & Hayman 1970). Ultimately for measuring the percentage of root colonization, the coloured roots were divided into the 1 cm fragments (Giovannetti & Moss 1980). The experiments were conducted in the statistical method of factorial design with the basis of randomized complete block samples in four replicates. The treatments were: (a) three levels of arbuscular mycorrhizal inoculants including non inoculation (F0), inoculation with Glomus etunicatum (F1) and Glomus intradices (F2), (b) four levels of Pseudomonas fluorescens inoculants including non inoculation (B0), inoculation with Pseudomonas fluorescens strains 4 (B0), Pseudomonas fluorescens strains 9 (B2), Pseudomonas fluorescens strains 12 (B3) and (c) two cultivars of sunflower including Euroflor (C1) and Master (C2). The analysis of variance was performed using SAS and Minitab softwares. Comparison of mean values was done using Duncan Multiple Range Test by MSTATC software at 5% probability level. Results and Discussion. The results of analysis of variance (ANOVA) on the basis of the major effect of fungus (F), bacterium (B) and cultivar (C) showed that the fungus (F) had significant effect on the fresh weight of above ground part, dry weight of root, fresh and dry weight of tray, tray diameter, leaf water potential, RWC, root colonization and leaf chlorophyll, while other indices did not affected by the fungus. In addition, the bacterium (B) had significant effect on all of the parameters except dry weight of root, leaf weight, leaf area, leaf water potential and leaf chlorophyll. Moreover, cultivar (C) significantly affected all of the parameters except leaf weight and RWC (Table 2). The comparison of mean for the major effect of fungus (F) showed that the F1 and F2 treatments had no significant difference with the control (F0) for the traits of fresh and dry weight of above ground part, leaf area and leaf weight. For the traits of dry weight of root, leaf water potential and leaf chlorophyll just the F2 treatment had significant difference with the control (F0). For the traits of fresh weight of tray, plant length and tray diameter just the F1 treatment had significant difference (p < 0.05) with the control (F0), while for the traits of dry weight of tray, root colonization and RWC both of the F1 and F2 treatments had significant differences (p < 0.05) with the control (F0). In related to comparison of mean for the major effect of bacterium (B), the B1 treatment significantly (p < 0.05) increased by itself the fresh weight of above ground part compared with the control (B0) treatment. A similar result was observed for the trait of dry weight of above ground part. Also, all of the bacterial treatments (B1, B2 and B3) significantly (p < 0.05) increased the fresh and dry weight of tray, tray diameter and RWC compared with the control treatment (B0). The B2 and B3 treatments significantly (p < 0.05) decreased the root colonization compared with the control treatment. For the trait of plant length, the comparison of mean showed that the B3 treatment significantly (p < 0.05) reduced this trait compared with the control and also, the maximum and minimum plant length were related to B1 and B3 treatments respectively. In addition the comparison of mean for the major effect of bacterium (B) on other characteristics such as dry weigh of roots, leaf chlorophyll, leaf water potential, leaf area and leaf weight did not showed significant differences. The comparison of mean for the major effect of cultivar (C) revealed that the C2 treatment (Master) had higher levels of fresh and dry weight of above ground part, dry weight of root, fresh and dry weight of tray, tray diameter, plant length, leaf water potential and root colonization than to the C1 treatment (Euroflor). Also, the C1 treatment had higher values of leaf area and chlorophyll than to the C2 treatment. The traits of leaf weight and RWC did not show significant differences between two cultivars (C1 and C2) of sunflowers (Table 3). The ANOVA for the interaction effect between fungus and bacterium (F × B) on the water relationships and agronomic indices of sunflower plants showed that this interaction was significant for all of the studied traits except fresh and dry weight of above ground part, dry weight of root and leaf chlorophyll (Table 2). Also the comparison of mean showed that, just the treatment of F2B1 had significant increasing difference (p <


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0.05) with the control treatment (F0B0) for the trait of fresh weight of above ground part. Similarly for the trait of dry weight of above ground part the treatments of F1B1 and F2B1 and for the trait of leaf water potential the treatments of F2B0 and F2B2 had significant difference (p < 0.05) with the control treatment (F0B0). In addition, all of the F × B treatments except F2B0 showed significant increasing difference with the control treatment (F0B0) for the traits of fresh weight of tray and tray diameter. The treatments of F0B3, F1B1, F1B3, F2B1 and F2B3 significantly increased the dry weight of tray compared with the control treatment (F0B0). Similarly, the treatments of F1B0, F1B1, F1B3, F2B0, F2B1 and F2B2 significantly increased the root colonization compared with the control treatment (F0B0). All of the F × B treatments had significant increasing difference (p < 0.05) with control treatment (F0B0) for the trait of RWC. Moreover, the treatment of F2B2 significantly reduced the plant length compared with the control treatment (Table 4).

Table 2

ANOVA for the effect of treatments on the water relationships and agronomic indices of sunflower plants (data are the mean squares)

S.O.V. df. Fresh weight of above ground

part

Dry weight of above

ground part

Dry weight of

root

Fresh weight of

tray

Dry weight of

tray Fungus (F) 2 227.188** 3.156ns 2.259** 50.619* 4.179*

Bacterium (B) 3 217.818** 12.211** 0.182ns 121.868** 3.094* Cultivar (C) 1 963.68** 191.507** 12.177** 313.168** 13.635**

F × B 6 56.202ns 1.398ns 0.132ns 71.594** 3.166** F × C 2 12.689ns 3.708ns 1.638* 28.314ns 6.949** B × C 3 153.957* 15.538** 0.847ns 60.685** 5.618** Error 72 42.633ns 1.997ns 0.406ns 14.875ns 0.899ns

S.O.V. df. Tray diameter Plant length Leaf weight Leaf area

Fungus (F) 2 0.55** 52.768ns 23.163ns 61209.885ns Bacterium (B) 3 0.6** 88.656** 9.462ns 22091.038ns Cultivar (C) 1 1.283** 7428.081** 1.862ns 129140.01*

F × B 6 0.316** 53.960* 33.526** 84480.288** F × C 2 0.192ns 6.142ns 4.021ns 10330.385ns B × C 3 0.087ns 76.078** 33.645* 84354.844* Error 72 0.081ns 17.755ns 8.478ns 21839.941ns

S.OV. df. Leaf water potential RWC Root colonization Leaf chlorophyll

Fungus (F) 2 0.236** 137.402** 1526.321** 10.345* Bacterium (B) 3 0.076ns 33.547** 205.930** 1.566ns Cultivar (C) 1 0.586** 8.766ns 298.144** 715.151**

F × B 6 0.135* 99.477** 145.700** 2.238ns F × C 2 0.099ns 0.137ns 32.422ns 1.599ns B × C 3 0.206** 22.132** 5.488ns 2.228ns Error 72 0.047ns 2.824ns 40.039ns 2.744ns

** and *: Significant at 1 and 5%, respectively and ns: Non significant The ANOVA for the interaction effect between fungus and cultivar (F × C) on the water relationships and agronomic indices of sunflower plants showed that, none of the studied traits except dry weight of root and tray did not affected by the interaction between fungus and cultivar (F × C) (Table 2). Also, the comparison of mean showed that between control treatments (F0C1 and F0C2), the F0C2 treatment significantly had higher levels of dry weight of above ground part and root, fresh and dry weight of tray, tray diameter, plant length and root colonization than to the F0C1 treatment, while F0C1


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treatment significantly had higher levels of leaf chlorophyll, leaf water potential and leaf area compared with the F0C2 treatment. Moreover, the F1C1 and F2C1 treatments had significant increasing difference (p < 0.05) with their control treatment (F0C1) for the traits of dry weight of above ground part and root, fresh and dry weight of tray and tray diameter. Similarly, the F1C2 and F2C2 treatments had significant increasing difference (p < 0.05) with their control treatment (F0C2) for the trait of leaf water potential. Furthermore, the F2C2 treatment had significant increasing difference (p < 0.05) with its control treatment (F0C2) for the trait of dry weight of root and leaf chlorophyll and both of the two cultivars (F1C1, F2C1, F1C2 and F2C2) significantly (p < 0.05) increased the root colonization and RWC compared with their control treatments (F2C1 and F0C2) (Table 5).

Table 3

The comparison of mean for the major effect of different levels of fungus, bacterium and cultivar on the water relationships and agronomic indices of sunflower plants (weight of

above ground parts and roots are in g)

Treatments Fresh weight of above ground

part

Dry weight of above

ground part

Dry weight of root

Fresh weight of tray

dry weight of tray

(F0) 85.948AB 14.58A 2.404B 35.689B 6.034B (F1) 83.968B 14.935A 2.514B 38.134A 6.741A (F2) 89.243A 15.206A 2.909A 37.422AB 6.521A (B0) 84.934B 14.503B 2.64A 33.915B 5.903B (B1) 90.573A 15.886A 2.634A 39.241A 6.681A (B2) 86.408B 14.966AB 2.681A 37.724A 6.626A (B3) 83.631B 14.273B 2.482A 37.448A 6.518A (C1) 83.218B 13.495B 2.253B 35.276B 6.055B (C2) 89.555A 16.319A 2.965A 38.888A 6.809A

Treatments Tray diameter (cm)

Plant length (cm)

Leaf weight (gr)

Leaf area (cm2)

(F0) 4.503B 57.398A 23.199AB 1175.22AB (F1) 4.762A 55.128B 22.103B 1117.88B (F2) 4.6B 57.303A 23.778A 1203.75A (B0) 4.388B 57.569AB 23.864A 1206.13A (B1) 4.667A 58.796A 23.148A 1171.33A (B2) 4.717A 55.506CB 22.611A 1145.58A (B3) 4.717A 54.569C 22.485A 1139.42A (C1) 4.506B 47.814B 23.166A 1202.29A (C2) 4.738A 65.406A 22.888A 1128.94B

Treatments Leaf water potential (– Bar)

RWC (%)

Root colonization (%)

Leaf chlorophyll (SPAD value)

(F0) 1.841A 74.220B 58.426B 43.488B (F1) 1.784A 77.502A 69.493A 43.747B (F2) 1.672B 78.053A 71.118A 44.576A (B0) 1.733A 74.885B 70.335A 43.869A (B1) 1.742A 77.149A 66.753AB 44.313A (B2) 1.738A 77.560A 64.155B 43.754A (B3) 1.850A 76.773A 64.140B 43.811A (C1) 1.688B 76.894A 64.583B 46.666A (C2) 1.844A 76.290A 68.108A 41.207B

Means, in each column, with at least one similar letters are not significantly different at the 5% probability level using Duncan multiple range test


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The ANOVA for the interaction effect between bacterium and cultivar (B × C) showed that, all of the traits related to water relationships and agronomic indices except dry weight of root, tray diameter, root colonization and leaf chlorophyll affected by the interaction between bacterium and cultivar (B × C) (Table 2). In addition, the comparison of mean showed that between control treatments (B0C1 and B0C2), the B0C2 treatment significantly had higher levels of fresh and dry weight of above ground part, dry weight of root, fresh and dry weight of tray, tray diameter, plant length and leaf weight than to the B0C1 treatment. Where as the treatment of B0C1 significantly showed higher levels of leaf chlorophyll, RWC and leaf water potential compared with the other control treatment (B0C2). Moreover in the Euroflor cultivar (C1), all of the bacterial treatments (B1C1, B2C1 and B3C1) had significant increasing difference (p < 0.05) with their control treatment (B0C1) for the traits of dry weight of above ground part, fresh weight of tray, tray diameter and RWC while, all of bacterial treatments (B1C1, B2C1 and B3C1) significantly reduce the leaf water potential. Similarly, the B1C1 and B2C1 treatments showed significant increasing difference (p < 0.05) with their control treatment (B0C1) for the trait of fresh weight of above ground part. Also, the B2C1 and B3C1 treatments had significant increasing difference (p < 0.05) with their control treatment (B0C1) for the traits of dry weight of tray and the B3C1 treatment significantly decreased root colonization compared with the control treatment (B0C1). However, in the Master cultivar (C2), the B1C2 treatment had significant increasing difference (p < 0.05) with its control treatment (B0C2) for the traits of fresh and dry weight of tray. Also, the B2C2 treatment had significant increasing and decreasing difference (p < 0.05) with its control treatment (B0C2) for the traits of leaf water potential and leaf area respectively. The B3C2 treatment had significant decreasing difference (p < 0.05) with its control treatment (B0C2) for the traits of fresh and dry weight of above ground part. A similar result was observed in the B2C2 and B3C2 treatments for the traits of root colonization and leaf weight. The B1C2 and B2C2 treatments had significant decreasing difference (p < 0.05) with their control treatment (B0C2) for the trait of plant length and also, the B1C2 and B2C2 treatments significantly increased tray diameter (Table 6).

Besides having difficulties for uptake of elements by plants, salinity can cause problems in water uptake. Increasing of soil salinity and therefore, enhancing of soluble salts concentration into the soil solution will leads to rising of osmotic pressure of soil solution. With increasing of osmotic pressure in soil solution, water uptake by roots will reduce and hence, leaf water potential and relative water content (RWC) will decrease. These disorders that occur in plant water relationships by salinity have a positive link with the plant photosynthesis and dry yield. Consequently, the problems of soil salinity for plant growth are almost similar those that caused by the drought stress (Blumwald 2000; Davies et al 2001). The results of this research confirmed that the inoculating of sunflower plants by AM fungi (Glomus etunicatum and Glomus intradices) can be so helpful in enhancing of leaf RWC. So that, the plants that were inoculated by Glomus intradices significantly showed higher levels of RWC than to the non-inoculated plants. The AM fungi with their complex networks of hyphae, can increase the available soil volume for roots. Hence, the plant available water and water relationships will improve at the presence of AM fungi and hereby plants can easily overcome to stresses caused by salt and drought (Hardie & Leyton 1981; Azcon 1987; Subramanian et al 2005). In the present research, the inoculated plants by Glomus intradices had higher levels of leaf chlorophyll compared with non-inoculated plants. It is shown that the rate of photosynthesis in mycorrhizal plants is more than non-mycorrhizal plants, which is probably due to the influence of this symbiosis on opening of leaf stomata because, closure of stomata limits gas exchange resulting in a decrease in photosynthesis (Wright et al 1998; Goss & de Varennes 2002; Sannazzaro et al 2006). The results of this study also showed that both of the AM fungi (Glomus etunicatum and Glomus intradices) are able to increase the root colonization and therefore, the sunflower plants may benefit from this important advantage. In addition, the Glomus etunicatum could slightly increase the dry weight of roots of sunflower plants. Other similar findings for increasing of dry weight of roots in mycorrhizal plants under saline condition were also reported before (Davies et al 2001). So that Feng et al (2002) believe that the higher


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accumulation of soluble sugars in mycorrhizal plant tissue, especially in roots, could make mycorrhizal plants more resistant to osmotic stress induced by exposure to salt.

Table 4 The comparison of mean for the interaction effect between fungus and bacterium on the water relationships and agronomic indices of sunflower plants (weight of above ground

parts and roots are in g)


part

Dry weight of above ground

part

Dry weight of root


dry weight of tray

(F0B0) 83.48BC 14.02CD 2.401A 30.85C 5.332C (F0B1) 88.56BC 15.42ABC 2.365A 36.54B 6.086ABC (F0B2) 88.60BC 14.67BCD 2.359A 36.99B 6.004BC (F0B3) 83.16BC 14.22CD 2.491A 38.38AB 6.715AB (F1B0) 81.59C 14.85ABCD 2.534A 39.48AB 7.066AB (F1B1) 86.77BC 15.92AB 2.575A 39.73AB 7.008AB (F1B2) 84.43BC 15.31ABC 2.665A 36.48B 6.696AB (F1B3) 83.08BC 13.66D 2.284A 36.84B 6.192ABC (F2B0) 89.74B 14.64BCD 2.985A 31.42C 5.310C (F2B1) 96.39A 16.32A 2.961A 41.45A 6.949AB (F2B2) 86.20BC 14.92ABCD 3.019A 39.70AB 7.178A (F2B3) 84.65BC 14.95ABCD 2.672A 37.12AB 6.646AB


Plant length (cm)

Leaf weight (gr)

Leaf area (cm2)

(F0B0) 4.112B 57.59ABC 23.87ABC 1205ABC (F0B1) 4.538A 59.28AB 23.79ABC 1205ABC (F0B2) 4.612A 57.47ABC 23.88ABC 1211ABC (F0B3) 4.750A 55.25BCD 21.26BC 1080BC (F1B0) 4.850A 54.41BCD 20.90BC 1057BC (F1B1) 4.738A 56.15ABCD 21.60BC 1091BC (F1B2) 4.725A 57.00ABC 23.45BC 1185BC (F1B3) 4.738A 52.95CD 22.47BC 1138BC (F2B0) 4.200B 60.70A 26.83A 1356A (F2B1) 4.725A 60.96A 24.05AB 1218AB (F2B2) 4.812A 52.05D 20.51C 1040C (F2B3) 4.662A 55.51BCD 23.73ABC 1201ABC


RWC (%)



(F0B0) 1.962A 66.63D 59.30DE 43.92AB (F0B1) 1.688BCDE 76.06C 60.69DE 43.83AB (F0B2) 1.812ABCD 77.57ABC 55.46E 43.20B (F0B3) 1.90AB 76.63C 58.26DE 42.99B (F1B0) 1.638CDE 78.80AB 74.21AB 43.07B (F1B1) 1.762ABCDE 76.22C 69.05BC 43.87AB (F1B2) 1.875ABC 77.87ABC 63.57CD 44.20AB (F1B3) 1.862ABC 77.12BC 71.13AB 43.84AB (F2B0) 1.600DE 79.23A 77.49A 44.61AB (F2B1) 1.775ABCD 79.17A 70.52AB 45.24A (F2B2) 1.525E 77.24BC 73.43AB 43.86AB (F2B3) 1.788ABCD 76.57C 63.04CD 44.60AB

Means, in each column, with at least one similar letters are not significantly different at the 5% probability level using Duncan multiple range test


46

With regarding to the role of AM fungi in facilitating of plant water relationships, increasing of plant yield at present of AM fungi is inevitable. Whereas in the greenhouse condition achievement to actual yield of sunflower plant in not possible therefore, the parameters that were related to yield of sunflower were used. One of these parameters is fresh and dry weight of tray. Our results revealed that both of the AM fungi (Glomus etunicatum and Glomus intradices) used in this study increased significantly the dry weight of tray. Also, the plants that were inoculated by Glomus etunicatum had higher fresh weigh of tray than to the none-inoculated plants. Jalaluddin (1993) observed that under salt stress, corn plants inoculated by Glomus intraradices showed a 400% increase in dry matter over control plants.

Table 5

The comparison of mean for the interaction effect between fungus and cultivar on the water relationships and agronomic indices of sunflower plants (weight of above ground

parts and roots are in g)


part

Dry weight of above

ground part

Dry weight of root


dry weight of tray

(F0C1) 83.45BC 12.78C 1.924C 32.80C 5.137B (F0C2) 88.45AB 16.38A 2.883B 38.58AB 6.932A (F1C1) 80.22C 13.78B 2.419B 36.77AB 6.742A (F1C2) 87.72AB 16.09A 2.609B 39.50A 6.739A (F2C1) 85.98B 13.92B 2.416B 36.25B 6.286A (F2C2) 92.50A 16.49A 3.403A 38.59AB 6.755A


Plant length (cm)

Leaf weight (gr)

Leaf area (cm2)

(F0C1) 4.30C 48.36B 23.74A 1232A (F0C2) 4.706AB 66.44A 22.66A 1118B (F1C1) 4.675AB 46.84B 21.97A 1140AB (F1C2) 4.85A 63.42A 22.24A 1096B (F2C1) 4.544B 48.24B 23.79A 1235A (F2C2) 4.656AB 66.36A 23.76A 1173AB


RWC (%)



(F0C1) 1.70BC 74.58B 55.71C 46.46A (F0C2) 1.981A 73.86B 61.15B 40.52C (F1C1) 1.750BC 77.82A 68.78A 46.43A (F1C2) 1.819B 77.18A 70.21A 41.06BC (F2C1) 1.612C 78.28A 69.27A 47.11A (F2C2) 1.731BC 77.82A 72.97A 42.04B

Means, in each column, with at least one similar letters are not significantly different at the 5% probability level using Duncan multiple range test One of the major effects of salinity on plants is the ethylene accumulation in their roots which decrease root growth and finally reduce the yield of crops. PGPRs those are able to produce ACC-deaminase in plants rhizosphere, can consume pre-produced ethylene (ACC) and convert it to α-ketobutyrate and ammonium, so they are able to reduce ethylenes level in plants and hence, increase their growth (Glick et al 1998; Penrose & Glick 2003). When the PGPR contains the enzyme ACC deaminase, the bacterial cells act as a sink for ACC, the immediate biosynthetic precursor of ethylene thereby lowering plant ethylene levels and decreasing the negative effects of various environmental stresses (Stearns et al 2005). The results of the present study showed that the plant inoculating by Pseudomonas fluorescence strain 4 increased the fresh and dry weight of


47

above ground part. Therefore, it seems that the Pseudomonas fluorescence strain 4 is able to increase plant biomass by reducing of ethylene level in sunflowers. All of the bacterial inoculants used in this study (Pseudomonas fluorescens strains 4, 9 and 12) significantly enhanced the fresh and dry weight of tray of sunflowers. Therefore, it is expected that with application of these bacteria, the yield of sunflower plants will increase per unit area under saline condition. Similar findings were pointed out in other plants grown under drought and salt stress at the present of PGPRs having the ability of producing ACC deaminase enzyme (Mayak et al 2003; Mayak et al 2004).

Table 6

The comparison of mean for the interaction effect between bacterium and cultivar on the water relationships and agronomic indices of sunflower plants (weight of above ground

parts and roots ar in g)

Treatments Fresh weight

of above ground part

Dry weight of above

ground part

Dry weight of root


dry weight of tray

(B0C1) 78.18D 12.16F 2.098C 31.14C 5.152C (B0C2) 91.68AB 16.84AB 3.182A 36.69B 6.653B (B1C1) 87.41BC 14.06DE 2.204C 35.71B 5.843BC (B1C2) 93.74A 17.71A 3.063AB 42.77A 7.518A (B2C1) 84.95C 14.12DE 2.582BC 37.16B 6.630B (B2C2) 87.87BC 15.82BC 2.780AB 38.28B 6.622B (B3C1) 82.33CD 13.63E 2.128C 37.09B 6.594B (B3C2) 84.93C 14.91CD 2.837AB 37.80B 6.442B


Plant length (cm)

Leaf weight (gr)

Leaf area (cm2)

(B0C1) 4.225C 46.37C 22.43B 1164ABC (B0C2) 4.55B 68.77A 25.30A 1248A (B1C1) 4.508B 49.83C 23.07AB 1197AB (B1C2) 4.825A 67.77A 23.23AB 1146ABC (B2C1) 4.683AB 48.44C 23.84AB 1237A (B2C2) 4.750AB 62.57B 21.38B 1054C (B3C1) 4.608AB 46.62C 23.33AB 1211A (B3C2) 4.825A 62.52B 21.46B 1068BC


RWC (%)



(B0C1) 1.525D 76.52A 68.54AB 46.73A (B0C2) 1.942A 73.25B 72.13A 41B (B1C1) 1.708C 76.49A 64.78BC 47.13A (B1C2) 1.775ABC 77.81A 68.73AB 41.49B (B2C1) 1.742BC 77.70A 63.07BC 46.71A (B2C2) 1.733BC 77.42A 65.24BC 40.80B (B3C1) 1.775ABC 76.88A 61.95C 46.09A (B3C2) 1.925AB 76.67A 66.33BC 41.53B

Means, in each column, with at least one similar letters are not significantly different at the 5% probability level using Duncan multiple range test With respecting to positive advantages of AM fungi and PGPRs for plants, it seems that with application of these two microorganisms simultaneously the plant growth will significantly improve under saline condition. So that, in the present study the co-inoculatation of sunflowers by Glomus intradices and Pseudomonas fluorescence strain 4 significantly increased their fresh and dry weight of above ground part compared with the control treatment. Also using of Glomus intradices alone did not increase significantly


48

fresh and dry weight of tray compared with the control, while addition of Pseudomonas fluorescence strain 4, 9 and 12 and AM fungi combination significantly increased these traits of sunflowers. In addition co-inoculation had significant effect on leaf water potential whereas, the traits of RWC, leaf chlorophyll and dry weight of root did not effect. Hence, the PGPRs with producing phytohormones and cohabiting in the rhizosphere with AM fungi could play a helper role in the plant-fungus interaction. However, Vázquez et al (2000) showed that that PGPRs like Pseudomonas did not exert an antagonistic effect against AM fungi in corn plants. Acknowledgements. We wish to thank University of Tehran for supporting field studies and samplings. We also would like to gratefully thank all the members of the soil science laboratory of faculty of agriculture, University of Tehran, for providing the facilities to carry out this work and for their suggestions. References Al-Karaki G. N., 2006 Nursery inoculation of tomato with arbuscular mycorrhizal fungi

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Received: 09 December 2009. Accepted: 29 December 2009. Published online: 31 December 2009. Authors: Mostafa Shirmardi, Department of Soil Science, Faculty of Water & Soil Engineering, University College of Agriculture & Natural Resources, University of Tehran, Karaj, 31587–77871, Iran. E-mail: [email protected] Gholam Reza Savaghebi, Department of Soil Science, Faculty of Water & Soil Engineering, University College of Agriculture & Natural Resources, University of Tehran, Karaj, 31587–77871, Iran. E-mail: [email protected] Kazem Khavazi, Soil & Water Research Institute, North Kargar Ave., Jalal-Al-Ahmad Highway, Tehran, 14155–6185, Iran. E-mail: [email protected] Ali Akbarzadeh, Department of Soil Science, Faculty of Water & Soil Engineering, University College of Agriculture & Natural Resources, University of Tehran, Karaj, 31587–77871, Iran. E-mail: [email protected] Mohsen Farahbakhsh, Department of Soil Science, Faculty of Water & Soil Engineering, University College of Agriculture & Natural Resources, University of Tehran, Karaj, 31587–77871, Iran. E-mail: [email protected] Farhad Rejali, Soil & Water Research Institute, North Kargar Ave., Jalal-Al-Ahmad Highway, Tehran, 14155–6185, Iran. E-mail: [email protected] Abdolvahab Sadat, Department of Soil Science, Faculty of Water & Soil Engineering, University College of Agriculture & Natural Resources, University of Tehran, Karaj, 31587–77871, Iran. E-mail: [email protected] How to cite this article: Shimardi M., Savaghebi G. R., Khavazi K., Akbarzadeh A., Farahbakhsh M., Rejali F., Sadat A., 2009 Water relationships and agronomic indices of sunflower infection by microbial inoculants under saline condition. AAB Bioflux 1(2):37-50.

aab bioflux · cv. pacovan) by an am fungi (glomus clarum) improved the dry weight of root (80%),...

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